RESUMO
The microenvironment of retinoblastoma, the solid malignancy of the developing retina, is immunosuppressive. To study the interactions between tumor-associated microglia/macrophages (TAMs) and tumor cells in retinoblastomas, we analyzed immunohistochemistry markers in 23 patient samples and characterized 105 secreted cytokines of 11 retinoblastoma cell models in culture. We detected profuse infiltration of CD163+ protumoral M2-like polarized TAMs in eyes enucleated due to cancer progression. Previous treatment of patients increased the number of TAMs but did not affect M2-like polarization. M2-like microglia/macrophages were almost absent in five eyes obtained from children enucleated due to nontumoral causes. CD8+ tumor-infiltrating lymphocytes (TILs) were moderately abundant in tumor eyes and very scarce in nontumoral ones. The expression of the immune checkpoint molecule PD-L1 was absent in 95% of the tumor samples, which is concordant with the finding of FOXP3+ Tregs infiltrating tumors. We confirmed the pathology results using single-cell transcriptome analysis of one tumor. We identified the cytokines extracellular matrix metalloproteinase inducer (EMMPRIN) and macrophage migration inhibitory factor (MIF), both with reported immunosuppressive activity, secreted at high levels in retinoblastoma primary cell cultures. Gene expression analysis of a large retinoblastoma cohort and single-cell transcriptome analysis confirmed that MIF and EMMPRIN were significantly upregulated in retinoblastomas, which led us to quantify both proteins by immunoassays in liquid biopsies (aqueous humor obtained from more than 20 retinoblastoma patients). We found a significant increase in the concentration of MIF and EMMPRIN in cancer patients, compared to 12 noncancer ones. Finally, we showed that macrophages derived from peripheral blood mononuclear cells increased the expression of markers of M2-like polarization upon exposure to retinoblastoma-conditioned medium or recombinant MIF. Overall, our findings suggest that retinoblastoma cell secretions induce the protumoral phenotype of this tumor. Our results might have clinical impact in the fields of biomarkers and treatment. © 2022 The Pathological Society of Great Britain and Ireland.
Assuntos
Neoplasias da Retina , Retinoblastoma , Humor Aquoso , Basigina , Humanos , Leucócitos Mononucleares , Neoplasias da Retina/genética , Secretoma , Microambiente TumoralRESUMO
Nanoparticle albumin-bound paclitaxel (nab-paclitaxel) shows potent preclinical anticancer activity in pediatric solid tumors such as Ewing sarcoma, rhabdomyosarcoma and neuroblastoma, but responses in clinical trials have been modest. In this work, we aimed to discover a rational biomarker-based approach to select the right candidate patients for this treatment. We assessed the efficacy of nab-paclitaxel in 27 patient-derived xenografts (PDX), including 14 Ewing sarcomas, five rhabdomyosarcomas and several other pediatric solid tumors. Response rate (partial or complete response) was remarkable in rhabdomyosarcomas (four of five) and Ewing sarcomas (four of 14). We addressed several predictive factors of response to nab-paclitaxel such as the expression of the secreted protein acidic and rich in cysteine (SPARC), chromosomal stability of cancer cells and expression of antiapoptotic members of the B-cell lymphoma-2 (Bcl-2) family of proteins such as Bcl-2, Bcl-xL, Bcl-W and Mcl-1. Protein (immunoblotting) and gene expression of SPARC correlated positively, while immunoblotting and immunohistochemistry expression of Bcl-2 correlated negatively with the efficacy of nab-paclitaxel in Ewing sarcoma PDX. The negative correlation of Bcl-2 immunoblotting signal and activity was especially robust (r = 0.8352; P = 0.0007; Pearson correlation). Consequently, we evaluated pharmacological strategies to inhibit Bcl-2 during nab-paclitaxel treatment. We observed that the Bcl-2 inhibitor venetoclax improved the activity of nab-paclitaxel in highly resistant Bcl-2-expressing Ewing sarcoma PDX. Overall, our results suggest that low Bcl-2 expression could be used to select patients with Ewing sarcoma sensitive to nab-paclitaxel, and Bcl-2 inhibitors could improve the activity of this drug in Bcl-2-expressing Ewing sarcoma.
Assuntos
Antineoplásicos , Neoplasias Ósseas , Rabdomiossarcoma , Sarcoma de Ewing , Criança , Humanos , Antineoplásicos/uso terapêutico , Biomarcadores , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/patologia , Osteonectina/genética , Osteonectina/metabolismo , Osteonectina/uso terapêutico , Paclitaxel/farmacologia , Paclitaxel/uso terapêutico , Rabdomiossarcoma/tratamento farmacológico , Sarcoma de Ewing/tratamento farmacológico , Sarcoma de Ewing/patologiaRESUMO
Fundoscopy is the standard method for diagnosis and follow-up of intraocular retinoblastoma, but it is sometimes insufficient to discern whether tumors are inactivated following treatments. In this work, we hypothesized that the amount of conserved nuclear DNA sequences in the cell-free DNA (cfDNA) fraction of the aqueous humor (AH) might complement fundoscopy for retinoblastoma follow-up. To address our hypothesis, we developed highly sensitive droplet digital polymerase chain reaction (ddPCR) methods to quantify highly conserved DNA sequences of nucleus-encoded genes (GAPDH and B4GALNT1) and of a mitochondrial gene, MT-ATP6. We obtained AH samples during intravitreal treatments. We analyzed 42 AH samples from 25 patients with intraocular retinoblastoma and 11 AH from controls (non-cancer patients). According to clinical criteria, we grouped patients as having progression-free or progressive retinoblastoma. cfDNA concentration in the AH was similar in both retinoblastoma groups. Copy counts for nucleus-derived sequences of GAPDH and B4GALNT1 were significantly higher in the AH from patients with progressive disease, compared to the AH from progression-free patients and control non-cancer patients. The presence of mitochondrial DNA in the AH explained that both retinoblastoma groups had similar cfDNA concentration in AH. The optimal cut-off point for discriminating between progressive and progression-free retinoblastomas was 108 GAPDH copies per reaction. Among patients having serial AH samples analyzed during their intravitreal chemotherapy, GAPDH copies were high and decreased below the cut-off point in those patients responding to chemotherapy. In contrast, one non-responder patient remained with values above the cut-off during follow-up, until enucleation. We conclude that the measurement of conserved nuclear gene sequences in AH allows follow-up of intraocular retinoblastoma during intravitreal treatment. The method is applicable to all patients and could be relevant for those in which fundoscopy evaluation is inconclusive.
Assuntos
Sequência de Bases , HumanosRESUMO
The goals of this work were to identify factors favoring patient-derived xenograft (PDX) engraftment and study the association between PDX engraftment and prognosis in pediatric patients with Ewing sarcoma, osteosarcoma, and rhabdomyosarcoma. We used immunodeficient mice to establish 30 subcutaneous PDX from patient tumor biopsies, with a successful engraftment rate of 44%. Age greater than 12 years and relapsed disease were patient factors associated with higher engraftment rate. Tumor type and biopsy location did not associate with engraftment. PDX models retained histology markers and most chromosomal aberrations of patient samples during successive passages in mice. Model treatment with irinotecan resulted in significant activity in 20 of the PDXs and replicated the response of rhabdomyosarcoma patients. Successive generations of PDXs responded similarly to irinotecan, demonstrating functional stability of these models. Importantly, out of 68 tumor samples from 51 patients with a median follow-up of 21.2 months, PDX engraftment from newly diagnosed patients was a prognostic factor significantly associated with poor outcome (p = 0.040). This association was not significant for relapsed patients. In the subgroup of patients with newly diagnosed Ewing sarcoma classified as standard risk, we found higher risk of relapse or refractory disease associated with those samples that produced stable PDX models (p = 0.0357). Overall, our study shows that PDX engraftment predicts worse outcome in newly diagnosed pediatric sarcoma patients.
Assuntos
Prognóstico , Sarcoma de Ewing/tratamento farmacológico , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Adolescente , Animais , Criança , Pré-Escolar , Modelos Animais de Doenças , Feminino , Xenoenxertos/efeitos dos fármacos , Humanos , Irinotecano/farmacologia , Irinotecano/uso terapêutico , Masculino , Camundongos , Osteossarcoma/tratamento farmacológico , Rabdomiossarcoma/tratamento farmacológico , Sarcoma/tratamento farmacológico , Resultado do TratamentoRESUMO
Ewing sarcoma is a bone and soft tissue tumor predominantly affecting adolescents and young adults. To characterize changes in anticancer drug activity and intratumor drug distribution during the evolution of Ewing sarcomas, we used immunodeficient mice to establish pairs of patient-derived xenografts (PDX) at early (initial diagnosis) and late (relapse or refractory progression) stages of the disease from three patients. Analysis of copy number alterations (CNA) in early passage PDX tissues showed that two tumor pairs established from patients which responded initially to therapy and relapsed more than one year later displayed similar CNAs at early and late stages. For these two patients, PDX established from late tumors were more resistant to chemotherapy (irinotecan) than early counterparts. In contrast, the tumor pair established at refractory progression showed highly dissimilar CNA profiles, and the pattern of response to chemotherapy was discordant with those of relapsed cases. In mice receiving irinotecan infusions, the level of SN-38 (active metabolite of irinotecan) in the intracellular tumor compartment was reduced in tumors at later stages compared to earlier tumors for those pairs bearing similar CNAs, suggesting that distribution of anticancer drug shifted toward the extracellular compartment during clonal tumor evolution. Overexpression of the drug transporter P-glycoprotein in late tumor was likely responsible for this shift in drug distribution in one of the cases.
Assuntos
Antineoplásicos , Neoplasias Ósseas , Preparações Farmacêuticas , Sarcoma de Ewing , Adolescente , Animais , Antineoplásicos/uso terapêutico , Neoplasias Ósseas/tratamento farmacológico , Humanos , Irinotecano , Camundongos , Sarcoma de Ewing/tratamento farmacológicoRESUMO
Retinoblastoma is a pediatric solid tumor of the retina activated upon homozygous inactivation of the tumor suppressor RB1 VCN-01 is an oncolytic adenovirus designed to replicate selectively in tumor cells with high abundance of free E2F-1, a consequence of a dysfunctional RB1 pathway. Thus, we reasoned that VCN-01 could provide targeted therapeutic activity against even chemoresistant retinoblastoma. In vitro, VCN-01 effectively killed patient-derived retinoblastoma models. In mice, intravitreous administration of VCN-01 in retinoblastoma xenografts induced tumor necrosis, improved ocular survival compared with standard-of-care chemotherapy, and prevented micrometastatic dissemination into the brain. In juvenile immunocompetent rabbits, VCN-01 did not replicate in retinas, induced minor local side effects, and only leaked slightly and for a short time into the blood. Initial phase 1 data in patients showed the feasibility of the administration of intravitreous VCN-01 and resulted in antitumor activity in retinoblastoma vitreous seeds and evidence of viral replication markers in tumor cells. The treatment caused local vitreous inflammation but no systemic complications. Thus, oncolytic adenoviruses targeting RB1 might provide a tumor-selective and chemotherapy-independent treatment option for retinoblastoma.
Assuntos
Adenoviridae/fisiologia , Terapia de Alvo Molecular , Vírus Oncolíticos/fisiologia , Proteína do Retinoblastoma/metabolismo , Retinoblastoma/metabolismo , Transdução de Sinais , Animais , Linhagem Celular Tumoral , Citotoxicidade Imunológica , Humanos , Camundongos , Metástase Neoplásica , Coelhos , Retinoblastoma/imunologia , Retinoblastoma/patologia , Análise de Sobrevida , Distribuição Tecidual , Pesquisa Translacional Biomédica , Resultado do Tratamento , Replicação Viral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We report for the first time on a nano-drug delivery system based on glucosylated polymeric nanomicelles to actively target the second-generation tyrosine kinase inhibitor dasatinib to glucose-avid pediatric sarcomas by the intravenous route. After a comprehensive physicochemical characterization that confirmed the substantially lower critical micellar concentration and the higher encapsulation capacity of the glucosylated amphiphilic nanocarrier with respect to the pristine counterpart, we showed a 9-fold decrease of the half maximal inhibitory concentration of dasatinib in a rhabdomyosarcoma cell line, Rh30, in vitro. In immunodeficient mice bearing the glucose-avid Rh30 xenograft, we revealed that the glucosylated polymeric nanomicelles increased the delivery of dasatinib in the tumor parenchyma. Conversely, the exposure of off-target tissues and organs to the drug was substantially reduced. Upon experimental confirmation that most patient-derived xenograft (PDX) models of pediatric sarcomas overexpress glucose transporter 1 (GLUT-1), we demonstrated the selective accumulation of dasatinib in a patient-derived rhabdomyosarcoma model in vivo. Conversely, the reference dose administered by the oral route was not tumor-selective. Finally, the improved nanocarrier pharmacokinetics led to prolonged median survival of mice bearing a clinically relevant PDX model of alveolar rhabdomyosarcoma from 19â¯days for the untreated controls to 27â¯days for the targeted therapy.
Assuntos
Antineoplásicos/administração & dosagem , Dasatinibe/administração & dosagem , Micelas , Nanoestruturas/administração & dosagem , Inibidores de Proteínas Quinases/administração & dosagem , Animais , Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Criança , Dasatinibe/farmacocinética , Glucose/metabolismo , Transportador de Glucose Tipo 1/genética , Transportador de Glucose Tipo 1/metabolismo , Transportador de Glucose Tipo 4/genética , Transportador de Glucose Tipo 4/metabolismo , Humanos , Camundongos Nus , Inibidores de Proteínas Quinases/farmacocinética , Sarcoma/tratamento farmacológico , Sarcoma/genética , Sarcoma/metabolismoRESUMO
Treatment of retinoblastoma -a pediatric cancer of the developing retina- might benefit from strategies to inhibit the blood-retinal barrier (BRB). The potent anticancer agent topotecan is a substrate of efflux transporters BCRP and P-gp, which are expressed at the BRB to restrict vitreous and retinal distribution of xenobiotics. In this work we have studied vitreous and retinal distribution, tumor accumulation and antitumor activity of topotecan, using pantoprazole as inhibitor of BCRP and P-gp. We used rabbit and mouse eyes as BRB models and patient-derived xenografts as retinoblastoma models. To validate the rabbit BRB model we stained BCRP and P-gp in the retinal vessels. Using intravitreous microdialysis we showed that the penetration of the rabbit vitreous by lactone topotecan increased significantly upon concomitant administration of pantoprazole (P=0.0285). Pantoprazole also increased topotecan penetration of the mouse vitreous, measured as the vitreous-to-plasma topotecan concentration ratio at the steady state (P=0.0246). Pantoprazole increased topotecan antitumor efficacy and intracellular penetration in retinoblastoma in vitro, but did not enhance intratumor drug distribution and survival in mice bearing the intraocular human tumor HSJD-RBT-2. Anatomical differences with the clinical setting likely limited our in vivo study, since xenografts were poorly vascularized masses that loaded most of the vitreous compartment. We conclude that pharmacological modulation of the BRB is feasible, enhances anticancer drug distribution into the vitreous and might have clinical implications in retinoblastoma. CHEMICAL COMPOUNDS INCLUDED IN THIS MANUSCRIPT: Topotecan (PubChem CID: 60700) Pantoprazole sodium (PubChem CID: 15008962).
Assuntos
2-Piridinilmetilsulfinilbenzimidazóis/farmacologia , Barreira Hematorretiniana/efeitos dos fármacos , Neoplasias da Retina/tratamento farmacológico , Retinoblastoma/tratamento farmacológico , Inibidores da Topoisomerase I/uso terapêutico , Topotecan/uso terapêutico , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Barreira Hematorretiniana/metabolismo , Humanos , Camundongos Nus , Pantoprazol , Coelhos , Neoplasias da Retina/genética , Neoplasias da Retina/metabolismo , Retinoblastoma/genética , Retinoblastoma/metabolismo , Inibidores da Topoisomerase I/farmacocinética , Topotecan/farmacocinética , Corpo Vítreo/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Neuroblastoma is a pediatric solid tumor with high expression of the tumor associated antigen disialoganglioside GD2. Despite initial response to induction therapy, nearly 50% of high-risk neuroblastomas recur because of chemoresistance. Here we encapsulated the topoisomerase-I inhibitor SN-38 in polymeric nanoparticles (NPs) surface-decorated with the anti-GD2 mouse mAb 3F8 at a mean density of seven antibody molecules per NP. The accumulation of drug-loaded NPs targeted with 3F8 versus with control antibody was monitored by microdialysis in patient-derived GD2-expressing neuroblastoma xenografts. We showed that the extent of tumor penetration by SN-38 was significantly higher in mice receiving the targeted nano-drug delivery system when compared to non-targeted system or free drug. This selective penetration of the tumor extracellular fluid translated into a strong anti-tumor effect prolonging survival of mice bearing GD2-high neuroblastomas in vivo.